Most clinically used antibiotics are natural products from microbial sources and are built of peptide or polyketide frameworks or hybrids thereof. In the biogenesis of peptide antibiotics of both ribosomal and nonribosomal origin one of Nature's strategies is to morph the amino acid side chains and also the peptide backbones into scaffolds that are conformationally restricted by cyclization to populate biologically active conformers. A complementary strategy is to generate functional groups within restricted architectures that lead to potent inhibition of specific targets. In this application we propose examination of both aspects to decipher enzymatic strategies for side chain and backbone cyclizations during peptide antibiotic scaffold maturation as well as generation of unusual functional groups that underlie antibiotic structure and function. In specific aim 1 we propose to decipher the enzymatic strategies for making stable N-P bonds in the ribosomal peptide antibiotic Microcin C7 and the nonribosomal peptide phaseolotoxin. In specific aim 2 we will examine the enzymatic machinery for construction of "syrbactin" antibiotics with 12 membered enamide macrolactam rings as conditional electrophiles that irreversibly target the proteasome. In specific aim 3 we examine antibiotic and pheromone synthases that convert the indole side chain of tryptophan to a rigidified tricyclic system as well as looking at the construction of an eight member macrocycle involving the indole ring. We also examine the enzymes that mediate conversion of Phe and Tyr side chains into rigidified bicyclic and tricyclic frameworks in toxin and antibiotic generation. PUBLIC HEALTH RELEVANCE: There is a constant need for new antibiotics as multiply drug resistant bacterial pathogens emerge. Most antibiotics are built on natural product scaffolds. This application examines sets of enzymes that build in conformational constraints and unusual side chains into natural peptide-based frameworks during antibiotic construction. These biosynthetic enzymes involve morphing of the amino acid building blocks, both in side chains and in peptide bond connectivity, to create the rigidified scaffolds with conditionally reactive functional groups that lead to biologically active conformers of antibiotics.